Multiple sclerosis (MS) is hypothesized to be an autoimmune disease where the activation of myelin-reactive T cells, possibly by cross-reactive antigens, mediates the initial inflammatory insult leading to the recruitment of effector inflammatory cells. While there are no differences in the frequency of MBP and PLP reactive T cells in MS patients as compared to controls using limiting dilution analysis (LDA), it has become clear that there are both functional differences in the myelin reactive T cells of MS patients and differences in their regulation. We recently developed methods to directly measure the frequency of clonally expanded and activated antigen specific and invariant T cells ex vivo without in vitro manipulations. This analysis uses PCR to measure the number of T cells expressing particular TCR alpha/beta chains. In contrast to frequencies of T cells recognizing the immunodominant MBPp85-99 epitope in MS patients of approximately 1:10/5 as measured by LDA, we found T cell frequencies as high as 1:300 in the same subjects by PCR analysis: This grant will focus on three major issues. First, we will investigate whether the clonal expansion we observed for MBPp85-99 reactive T cells is also observed for T cells recognizing other immunodominant regions of MBP and PLP. This is critical to known in the development of antigen specific immunotherapies. As autoreactive T cells are present in the circulation of normal individuals, it has also become clear that other populations of T cells exist which may regulate the activation of autoreactive T cells, including a recently described subset of T cells expressing the invariant Valpha24JalphaQ sequences. We recently demonstrated that the Valpha24JalphaQ T cells are reduced ion frequency and are Th1 like (lacking IL-4 secretion) in diabetic twins as compared to non-diabetic twins. As the same methods can be used to quantify the frequency of Valpha24JalphaQ in patients with MS. The third aim of the grant will focus on the loss of IL-4 producing CD4+/B7.1+ cells, we recently observed in patients with MS. Using the PCR/TCR technology to identify antigen reactive T cells, we will explore the hypothesis that MS patients have a loss of myelin reactive T cells in the ex vivo IL-4 producing population, while in normal subjects IL-4 producing T cells are autoreactive. These experiments will provide insights that may be important for the design of antigen specific immunotherapies for patients with multiple sclerosis.